Clostridium tetani toxin
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1 Clostridium tetani toxin IgG - ELISA Enzyme immunoassay for the quantitative determination of IgG-class antibodies against Clostridium tetani toxin in human serum or plasma For Research Use Only GenWay Biotech, Inc Nancy Ridge Drive San Diego, CA Phone: Fax: Product Number: (96 Determinations)
2 1. INTRODUCTION Clostridia are spore-forming gram-positive bacteria. The round spores are build at the terminal end which results in the microscope in a tennis racket like shape. Tetanus develops only when spores of Clostridium tetani germinate under strict anaerobic conditions after gaining access to wounds and small lacerations. The clinical manifestation of the disease is primary not caused by the invasion of the exciter but by the secretion of a powerful neurotoxin (tetanospasmin). This toxin blocks the inhibition of the signal transduction and has a high affinity to the central nervous system. The consequence is hyper excitability of the muscles to external stimuli in combination with a principal increase of the muscle tonus without influence of consciousness. It starts with tonic spasm of muscles (trimus), mimic muscles and gallet muscles. Neck, back and abdominal musculature follow. At the same time the appearance of refectory spasm of whole muscle groups can hamper breathing. Hyper salivation and swallowing problems cause aspiration and pneumonia with the next breath. Clostridium tetani is ubiquitous present in soil and intestine of humans and animals. Ingestion of bacteria or growth in the intestine of man or animal is without harm. The spores are extremely resistant towards heat and can stay infectious for a long period. The bacteria can get under the skin by even smallest wounds. In Europe tetanus mainly occurs after injuries and sometimes postoperative whereas in developing countries Tetanus is widely disseminated. The WHO assumes that one million people die because of tetanus worldwide per year. Tetanus toxin is an excellent immunogen in man - only one antigenic type of toxin. The only effective way to control tetanus is by prophylactic active immunization. Species Disease Symptoms Mechanism of infection Clostridium tetani Tetanus Severe and painful spasms and rigidity of voluntary muscles, followed by cardiac, renal and circulatory failure Injury, inoculation Infection may be identified by Microscopy: Gram stain Serology: Detection of toxin production by ELISA 2. INTENDED USE The GenWay Clostridium tetani toxin IgG-ELISA is intended for the quantitative determination of IgG class antibodies against Clostridium tetani toxin in human serum or plasma (citrate). This allows for the determination of the immune status of the patients facilitating individual recommendations about the necessity of a basic immunization or booster injection. 3. PRINCIPLE OF THE ASSAY The quantitative immunoenzymatic determination of IgG-class antibodies against Tetanus toxoid is based on the ELISA (Enzyme-linked Immunosorbent Assay) technique. Microtiter strip wells are precoated with inactivated Tetanus toxoid antigens to bind coresponding antibodies of the specimen. After washing the wells to remove all unbound sample material horseradish peroxidase (HRP) labelled anti-human IgG conjugate is added. This conjugate binds to the captured Tetanus toxoid specific antibodies. The immune complex formed by the bound conjugate is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is proportional to the amount of toxoid specific IgG antibodies in the specimen. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450 nm is read using an ELISA microwell plate reader. 4. MATERIALS 4.1 Reagents supplied Clostridium tetani toxin Coated Wells (IgG): 12 breakapart 8-well snap-off strips coated with inactivated Tetanus toxoid antigen; in resealable aluminium foil. IgG Sample Diluent ***: 1 bottle containing 100 ml of buffer for sample dilution; ph 7.2 ± 0.2, coloured yellow; ready to use; white cap. Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.2 mol/l; ready to use; red cap. Washing Solution (20x conc.)*: 1 bottle containing 50 ml of a 20-fold concentrated buffer for washing the wells; ph 7.2 ± 0.2; white cap. Clostridium tetani toxin anti-igg Conjugate**: 1 bottle containing 20 ml of peroxidase labelled antibodies to human IgG; coloured blue; ready to use; black cap. TMB Substrate Solution: 1 bottle containing 15 ml 3,3',5,5'-tetramethylbenzidine (TMB); ready to use; yellow cap. Clostridium tetani toxin IgG s***: 4 vials, each containing 2ml; ready to use: A: 0.0 IU/ml; blue cap B: 0.1 IU/ml; green cap C: 0.5 IU/ml; yellow cap D: 1.0 IU/ml; red cap * contains 0.1 % Bronidox L after dilution ** contains 0.2 % Bronidox L *** contains 0.1 % Kathon 4.2. Materials supplied 1 Strip holder 1 Cover foil 1 Test protocol 1 distribution and identification plan 4.3. Materials and Equipment needed ELISA microwell plate reader, equipped for the measurement of absorbance at 450/620nm Incubator 37 C 2
3 Manual or automatic equipment for rinsing wells Pipettes to deliver volumes between 10 and 1000 µl Vortex tube mixer Deionised or (freshly) distilled water Disposable tubes Timer 5. STABILITY AND STORAGE The reagents are stable up to the expiry date stated on the label when stored at C. 6. REAGENT PREPARATION It is very important to bring all reagents, samples and controls to room temperature (20 25 C) before starting the test run! 6.1. Coated Snap-off Strips The ready to use breakapart snap-off strips are coated with inactivated Tetanus toxoid antigens. Store at C. Immediately after removal of strips, the remaining strips should be resealed in the aluminium foil along with the desiccant supplied and stored at C; stability until expiry date Clostridium tetani toxin anti-igg Conjugate The bottle contains 20 ml of a solution with anti human IgG horseradish peroxidase, buffer, stabilizers, preservatives and an inert blue dye. The solution is ready to use. Store at C. After first opening stability until expiry date when stored at 2 8 C s The vials labelled with A, B, C and D contain a ready to use standard solution. The concentration of the standards, calibrated in accordance with the 1st International for TETANUS IMMUNOGLOBULIN, HUMAN (Code TE-3) of the National Institute for Biological s and Control (NIBSC), Potters Bar, UK, are: A: 0.0 IU/ml B: 0.1 IU/ml C: 0.5 IU/ml D: 1.0 IU/ml The solutions have to be stored at C and contain 0.1% Kathon. After first opening stability until expiry date when stored at 2 8 C IgG Sample Diluent The bottle contains 100 ml phosphate buffer, stabilizers, preservatives and an inert yellow dye. It is used for the dilution of the patient specimen. This ready to use solution has to be stored at 2 8 C. After first opening stability until expiry date when stored at 2 8 C Washing Solution (20xconc.) The bottle contains 50 ml of a concentrated buffer, detergents and preservatives. Dilute washing solution 1+19; e.g. 10 ml washing solution ml fresh and germ free redistilled water. The diluted buffer will keep for 5 days if stored at room temperature. Crystals in the solution disappear by warming up to 37 C in a water bath. After first opening stability until expiry date when stored at 2 8 C TMB Substrate Solution The bottle contains 15 ml of a tetramethylbenzidine/hydrogen peroxide system. The reagent is ready to use and has to be stored at C, away from the light. The solution should be colourless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away. After first opening stability until expiry date when stored at 2 8 C Stop Solution The bottle contains 15 ml 0.2 M sulphuric acid solution (R 36/38, S 26). This ready to use solution has to be stored at 2 8 C. After first opening stability until expiry date. 7. SPECIMEN COLLECTION AND PREPARATION Use human serum or plasma (citrate) samples with this assay. If the assay is performed within 5 days after sample collection, the specimen should be kept at C; otherwise they should be aliquoted and stored deep-frozen (-20 to -70 C). If samples are stored frozen, mix thawed samples well before testing. Avoid repeated freezing and thawing. Heat inactivation of samples is not recommended Sample Dilution Before assaying, all samples should be diluted with IgG Sample Diluent. Dispense 10µl sample and 1ml IgG Sample Diluent into tubes to obtain a dilution and thoroughly mix with a Vortex. For patients with expected antitoxin concentrations greater than D (1.0 IU/ml) a second dilution of this diluted patient sample should be performed; e.g. 100 µl of first sample dilution µl of IgG Sample Diluent (mix well). Dilution factor: 2 8. ASSAY PROCEDURE 8.1. Test Preparation Please read the test protocol carefully before performing the assay. Result reliability depends on strict adherence to the test protocol as described. The following test procedure is only validated for manual procedure. If performing the test on ELISA automatic systems we recommend to increase the washing steps from three to five and the volume of washing solution from 300µl to 350µl to avoid washing effects. Prior to commencing the assay, the distribution and identification plan for all specimens and controls should be carefully established on the result sheet supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder. 3
4 Please allocate at least: 1 well (e.g. A1) for the substrate blank, 4 wells (e.g. B1, C1, etc.) for A, B, C and D. It is recommended to determine patient samples in duplicate. Perform all assay steps in the order given and without any appreciable delays between the steps. A clean, disposable tip should be used for dispensing each control and sample. Adjust the incubator to 37 ± 1 C. 1. Dispense 100µl of each (A, B, C and D) and diluted samples into the respective wells. Leave well A1 for substrate blank. 2. Cover wells with the foil supplied in the kit. 3. Incubate for 1 hour ± 5 min at 37±1 C. 4. When incubation has been completed, remove the foil, aspirate the content off the wells and wash each well three times with 300µl of washing solution. Avoid overflows from the reaction wells. The soak time between each wash cycle should be >5 sec. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step! Note: Washing is critical! Insufficient washing results in poor precision and falsely elevated absorbance values. 5. Dispense 100µl Tetanus toxoid anti-igg Conjugate into all wells except for the blank well (e.g. A1). Cover with foil. 6. Incubate for 30 min at room temperature (20 to 25 C). Do not expose to direct sunlight. 7. Repeat step Dispense 100µl TMB Substrate Solution into all wells 9. Incubate for exactly 15 min at room temperature (20 to 25 C) in the dark. 10. Dispense 100µl Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution. Any blue colour developed during the incubation turns into yellow. Note: Highly positive patient samples can cause dark precipitates of the chromogen! These precipitates have an influence when reading the optical density. Predilution of the sample - as described under 7.1.Sample Dilution - is recommended. 11. Measure the absorbance of the specimen at 450/620nm within 30 min after addition of the Stop Solution Measurement Adjust the ELISA Microwell Plate Reader to zero using the substrate blank in well A1. If - due to technical reasons - the ELISA reader cannot be adjusted to zero using the substrate blank in well A1, subtract the absorbance value of well A1 from all other absorbance values measured in order to obtain reliable results! Measure the absorbance of all wells at 450 nm and record the absorbance values for each control and patient sample in the distribution and identification plan. Dual wavelength reading using 620 nm as reference wavelength is recommended. Where applicable calculate the mean absorbance values of all duplicates. 9. RESULTS 9.1. Assay Validation Criteria In order for an assay to be considered valid, the following criteria must be met: Substrate blank in A1: Absorbance < A in B1: Absorbance < B in C1: Absorbance > C in D1: Absorbance > D in E1: Absorbance > A < B < C < D If these criteria are not met, the test is not valid and must be repeated Calculation of Results In order to obtain quantitative results in IU/ml plot the (mean) absorbance values of 4 standards A, B, C and D on (linear/linear) graph paper in a system of coordinates against their corresponding concentrations (0.0 / 0.1 / 0.5 and 1.0 IU/ml) and draw a standard calibration curve (absorbance values on the vertical y- axis, concentrations on the horizontal x-axis). Read results (antitoxin IgG concentrations in IU/ml) from this standard curve employing the (mean) absorbance values of each patient specimen and control. Note: Readings of additionally (1+1) diluted patient samples must be multiplied by the appropriate dilution factor in order to obtain correct results! (Dilution: 1+1 = Dilution factor: 2). All suitable computer programs available can be used for automated result reading and calculation. 4
5 9.3. Typical Calibration Curve 9.4. Interpretation of Results and Recommendations [IU/ml] < 0.01 No protective antibody level! Immediate full course of basic immunization is recommended! No reliable protection! Booster injection and control of antibody concentration 4 to 6 weeks later is recommended Reliable protection! Booster injection and control of antibody concentration 4 to 6 weeks later is recommended Reliable protection; control of antibody concentration after about 2 years is recommended. Booster injection is not required. Note: In cases of antibody concentrations greater than 0.5 IU/ml vaccination can cause side effects! Range of long term protection: Control after 5 to 10 years. > 5.0 Range of long term protection: Control after 10 years. 10. SPECIFIC PERFORMANCE CHARACTERISTICS Precision Intraassay n Mean CV (%) Weak pos. Serum Pos. Serum Interassay n Mean (IU/ml) CV (%) Weak pos. Serum Pos. Serum Diagnostic Specificity The diagnostic specificity is defined as the probability of the assay of scoring negative in the absence of the specific analyte. It is >95 % Diagnostic Sensitivity The diagnostic sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific analyte. It is >95 % Analytical Sensitivity The analytical sensitivity defined as the apparent concentration of the analyte that can be distinguished from the zero calibrator is <0.05 IU/ml Interferences Interferences with hemolytic, lipemic or icteric sera are not observed up to a concentration of 10 mg/ml hemoglobin, 5 mg/ml triglycerides and 0.2 mg/ml bilirubin. Note: The results refer to the groups of samples investigated; these are not guaranteed specifications. 11. LIMITATIONS OF THE PROCEDURE Bacterial contamination or repeated freeze-thaw cycles of the specimen may affect the absorbance values. Diagnosis of an infectious disease should not be established on the basis of a single test result. A precise diagnosis should take into consideration clinical history, symptomatology as well as serological data. In immunocompromized patients and newborns serological data only have restricted value. 5
6 12. PRECAUTIONS AND WARNINGS In compliance with article 1 paragraph 2b European directive 98/79/EC the use of the in vitro diagnostic medical devices is intended by the manufacturer to secure suitability, performances and safety of the product. Therefore the test procedure, the information, the precautions and warnings in the instructions for use have to be strictly followed. The use of the testkits with analyzers and similar equipment has to be validated. Any change in design, composition and test procedure as well as for any use in combination with other products not approved by the manufacturer is not authorized; the user himself is responsible for such changes. The manufacturer is not liable for false results and incidents for these reasons. The manufacturer is not liable for any results by visual analysis of the patient samples. Only for in-vitro diagnostic use. All components of human origin used for the production of these reagents have been tested for anti-hiv antibodies, anti-hcv antibodies and HBsAg and have been found to be non-reactive. Nevertheless, all materials should still be regarded and handled as potentially infectious. Do not interchange reagents or strips of different production lots. No reagents of other manufacturers should be used along with reagents of this test kit. Do not use reagents after expiry date stated on the label. Use only clean pipette tips, dispensers, and lab ware. Do not interchange screw caps of reagent vials to avoid cross-contamination. Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination. After first opening and subsequent storage check conjugate and control vials for microbial contamination prior to further use. To avoid cross-contamination and falsely elevated results pipette patient samples and dispense conjugate without splashing accurately to the bottom of wells. The GenWay ELISA is only designed for qualified personnel who are familiar with good laboratory practice. WARNING: WARNING: In the used concentration Bronidox L has hardly any toxicological risk upon contact with skin and mucous membranes! Sulphuric acid irritates eyes and skin. Keep out of the reach of children. Upon contact with the eyes, rinse thoroughly with water and consult a doctor! Disposal Considerations Residues of chemicals and preparations are generally considered as hazardous waste. The disposal of this kind of waste is regulated through national and regional laws and regulations. Contact your local authorities or waste management companies which will give advice on how to dispose hazardous waste. 13. ORDERING INFORMATION Prod. No.: Clostridium tetani toxin IgG ELISA (96 Determinations) BIBLIOGRAPHY Ambrosch,F.,Wiedermann,G.,Müller,H.: Eine neue Mikro-ELISA-Methode zur Bestimmung der Tetanus- Antikörper.Zbl.Bakt.Hyg.A258, (1984) Mai,K.,Bartelheimer,H.K.,Rosin,H.:Über den Stand und die Dauer des Impfschutzes gegentetanus bei Kindern.Dtsch.med.Wschr.95,1044 (1970) Müller,H.E.,Müller,M.,Schiek,W.: Tetanus-Schutzimpfung-Indikation und Kontraindikation.Dtsch.med.Wschr.113,1326 (1988) Pietsch,M.:Impfserologie zur Ergänzung von Impfungen.Allgemeinarzt 15, (1993) Schröder,J.P.,Kuhlmann,W.D.: Tetanusimmunität bei Männern und Frauen in der BRD.Immun.Infekt.19,14 (1991 6
7 SCHEME OF THE ASSAY Clostridium tetani toxin IgG ELISA Assay Preparation Prepare reagents and samples as described. Establish the distribution and identification plan for all specimens and controls on the result sheet supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder. Substrate blank (z.b. A1) Assay Procedure A B C D Sample (diluted 1+100) A - 100µl B µl C µl - - D µl - Sample (diluted 1+100) µl Cover wells with foil supplied in the kit Incubate for 1 h at 37 C Wash each well three times with 300µl of washing solution Conjugate - 100µl 100µl 100µl 100µl 100µl Cover wells with foil supplied in the kit Incubate for 30 min at room temperature Wash each well three times with 300µl of washing solution TMB Substrate 100µl 100µl 100µl 100µl 100µl 100µl Incubate for15 min at room temperature in the dark Stop Solution 100µl 100µl 100µl 100µl 100µl 100µl Photometric measurement at 450 nm (reference wavelength: 620 nm) engl cr 7
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